Complex and evolving mechanisms of insecticide resistance in malaria vectors Malaria is a major public health issue in endemic countries like Mali. Insecticide treated bed net (ITN) campaigns have been very successful in reducing transmission, but the long term effectiveness of ITNs is being threatened by the complex and rapid evolution of insecticide resistance in the major vector species, like A. coluzzii. For example, we recently described adaptive introgression as a mechanism by which A. coluzzii acquired resistance. In this case the well-- characterized target--site resistance allele, kdr-, was transferred from the sister species A. gambiae into A. coluzzii via hybridization. Insecticide resistance in A. coluzzii also likely involves increased activity of metabolic genes like CYP9K1, a P450 cytochrome monooxygenase gene. CYP9K1 has been associated with resistance in two gene expression studies. Furthermore, our preliminary results show evidence for selection on cis-regulatory variation and increased copy number at CYP9K1 since the start of an ITN campaign in 2006. A. coluzzii has significantly increased in proportion in the population since 2006, suggesting that kdr-w and alleles at CYP9K1 confer a relative fitness advantage. In this study, we will examine the relative effects of an introgressed coding mutation (kdr-w), cis-regulatory variation at CYP9K1, and increased copy number of CYP9K1 on gene expression and insecticide resistance in A. coluzzii. Improving our understanding of the complex ways in which vector mosquitoes adapt to insecticide exposure will inform strategies aimed at managing resistance. For example, our results on the contribution of CYP9K1 to insecticide resistance may support the usefulness of adding PBO, a P450 inhibitor, to insecticide treated bed nets.